The goals of this proposal are to define the molecular mechanisms involved in vertebrate primary mouth formation, using the frog Xenopus tropicalis as a model. The primary mouth (also called the stomodeum, buccopharyngeal membrane) is the initial opening from the outside of the embryo to the gut. Primary mouth formation is an essential step in cranoiofacial development, but is not well understood. In vertebrates, the neural crest grows around the primary mouth to form the jaws and face and a secondary mouth opening must therefore form. The primary mouth then becomes the pharyngeal opening. The primary mouth forms from a unique region at the extreme anterior of the embryo where ectoderm and endoderm are directly juxtaposed. In order to define the molecular mechanisms involved in this process, we began to characterize this process in X. laevis. We fate mapped the presumptive primary mouth, and defined tissues required for its induction. In order to define genes required for primary mouth formation, we used database mining and expression microarray analysis, to identify genes whose expression is enriched in the presumptive primary mouth region. Two genes that came out of this analysis are fgf8 and the Wnt pathway inhibitor, frzb1, and preliminary data indicates that both are essential for primary mouth formation. We propose to extend this project to X. tropicalis. Compared to X. laevis, this species has the advantage of a diploid genome, making antisense knockdowns more effective, and offering the ability to rapidly prepare stable transgenic lines. Initial data suggests that primary mouth formation in X. laevis and X. tropicalis is very similar. I hypothesize that the process of primary mouth formation is highly conserved. I further hypothesize that primary mouth formation requires the coordinate and sequential input of multiple regulatory genes expressed from several inducing regions. We will examine the temporal and spatial requirement for Fgf and Frzbl function during primary mouth formation using transgenic lines expressing inducible dominant negative or RNAi constructs, and transplant assays. We will begin to examine the requirement for other genes using antisense approaches. This proposal is exploratory since the study is new and unpublished, since it proposes extension into a new species and since it requires reagent development. Reagents developed will be important not only for this project, but for the X. tropicalis community. The primary mouth is an essential structure required for normal eating and speaking. Craniofacial development is severely impacted by an abnormal or absent primary mouth, however the molecular mechanisms underlying this process are not understood. This study will add insight into the genetic basis of craniofacial abnormalities, focusing on a crucial structure. [unreadable] [unreadable] [unreadable]